This invention relates to base sequences capable of directing the expression of therapeutic genes specifically to squamous epithelia, and to medicaments for gene therapy capable of directing the expression of therapeutic genes specifically to squamous epithelia by the use of the base sequences.
Gene therapy is a therapeutic method by which genes or cells having the introduced genes are administered to the human body for the purpose of the treatment of diseases, and it has drawn attention as a new modality which may replace those currently in use. In 1990 gene therapy was performed on two children who had congenial severe combined immunodeficiency resulting from adenosine deaminase (ADA) deficiency in the United States. Since then over 2,100 cases of gene therapy had been performed around the world till the end of 1996. In this country, the first gene therapy on an ADA deficient patient was initiated in August of 1995.
In the research of gene therapy an indispensable technique is to devise an improvement in the safety of medicaments for gene therapy; for example, targeting with which therapeutic genes are only introduced (transferred) into cells that are the subject of treatment not only increases the effectiveness by preventing the administered vector for gene therapy from uselessly diffusing, but also decreases the expression of exogenous genes in unwanted organs or tissues, which then makes it an indispensable technique.
Specifically, there are techniques disclosed among others: a HIV vector that is patterned after human immunodeficiency virus (HIV) utilizes the propensity of its envelope protein (existing on the surface) specifically binding to CD4 protein, and can transfer genes only into CD4 positive cells (Shimada T. et al., J. Clin. Invest., 88, 1043, 1991); a recombinant viral vector derived by modifying the envelope of Moloney murine leukemia virus with heregulin can transfer genes only into cells such as lung cancer cells that overexpress epidermal growth factor receptor (EGFR) (Han X. L., et al., Proc. Natl. Acad. Sci. USA, 92, 21, 9747, 1995); a folate conjugated to poly-L-lysine having enhanced affinity to cancer cells which relies on the fact that the folate receptor is overexpressd in cancer cells (Gottschalk S., et al., Gene Ther., 1, 3, 185, 1994; Lee R. J., Hung L., J. Biol. Chem., 271, 14, 8481, 1996).
In the meantime, the development of a system capable of directing the expression of a gene specifically to a tissue which is the subject of treatment (tissue specific expression system) is under way. The tissue specific expression system is a technique that focuses on a protein which is only expressed in a certain organ or tissue and that utilizes a gene sequence regulating the expression of a gene such as a promoter and/or an enhancer for the gene that encode this protein.
This technique has been disclosed in reviews that are already in public knowledge (e.g., Gabi U. Dachs, et al., Oncology Res., 9, 313, 1997). The tissue-specific promoters/enhancers which are known include the promoter/enhancer for xcex1-fetoprotein (AFP) the specific expression of which can be seen in hepatocellular carcinoma, the promoter region of prostate specific antigen (PSA) the expression of which is increased in prostate cancer, the von Willebrand factor (vWf) and the tie-2/tek promoter both of which are endothelial cell specific, the DF3 promoter the increased expression of which can be seen in breast cancer, the albumin enhancer that is expressed specifically in liver, the tyrosinase promoter that can be specifically expressed in melanoma cells, the myellin basic protein (MBP) promoter that is specific for glioma cells, the osteocalcin promoter that is specific for osteosarcoma cells, and the like.
The disease-specific promoters/enhancers include the promoter for carcinoembryonic antigen (CEA) the increased expression of which can be seen in many carcinomas, the HER/neu promoter exhibiting enhanced expression in breast and pancreatic cancers, and the Myc-Max response element that activates transcription by the action of the Myc protein family/Max protein complex which is implicated in cell proliferation, differentiation, and apoptosis. The promoters/enhancers the expression of which is controlled by a wide variety of conditions have also been reported, including the promoter for early growth response-1 gene (Egr-1) upon radiation, tissue type plasminogen activator (t-PA), the GRP78/BiP protein promoter that is induced by tumor-specific conditions such as glucose deprivation and anoxia, hypoxia response element (HRE) exhibiting specific expression under hypoxic conditions, and the like.
However, against squamous cell carcinomas such as uterine cervical cancer, skin cancer, head and neck cancer, esophagus cancer, and lung cancer which are reported to account for about 60% of all the carcinomas, there has been no system capable of directing the expression of genes specifically to those carcinomas: there has been no system for carrying out effective gene therapy on these squamous cell carcinomas.
It is an object of this invention to provide a base sequence capable of directing the expression of a therapeutic gene specifically to squamous epithelium. It is also an object of the invention to provide a medicament for gene therapy capable of directing the expression of a therapeutic gene specifically to squamous epithelium by the use of said base sequence.
As a result of having pursued diligent investigations on the above-stated problems, the present inventors found novel base sequences capable of directing the expression of therapeutic genes specifically to squamous epithelia. Further, it was found that these novel base sequences had more enhanced capability of gene expression in squamous cell carcinomas such as SKG IIIa cells than in normal squamous epithelia; and this led to the completion of this invention.
Specifically, this invention relates to a base sequence for the expression of a therapeutic gene, said sequence capable of directing the expression of the therapeutic gene specifically to squamous epithelium.
More specifically, it relates to a base sequence for the expression of a therapeutic gene, said sequence being shown in any one of from (a) to (d) and capable of directing the expression of a therapeutic gene specifically to squamous epithelium:
(a) the base sequence set forth in SEQ ID NO:1 in the Sequence Listing;
(b) the base sequence set forth in SEQ ID NO:18 in the Sequence Listing;
(c) the base sequence set forth in SEQ ID NO:17 in the Sequence Listing; and
(d) the base sequence set forth in SEQ ID NO:16 in the Sequence Listing.
Also, this invention relates to a base sequence for the expression of a therapeutic gene, said sequence being shown in any one of from (a) to (d) in which one or more bases have been deleted or substituted, or to which one or more bases have been added and capable of directing the expression of a therapeutic gene specifically to squamous epithelium:
(a) the base sequence set forth in SEQ NO:1 in the Sequence Listing;
(b) the base sequence set forth in SEQ NO:18 in the Sequence Listing;
(c) the base sequence set forth in SEQ ID NO:17 in the Sequence Listing; and
(d) the base sequence set forth in SEQ ID:16 in the Sequence Listing.
Further, this invention relates to a medicament for gene therapy having the base sequence for the expression of a therapeutic gene as described above and the therapeutic gene in the downstream thereof, said medicament capable of directing the expression of a therapeutic gene specifically to squamous epithelium. Still further, it encompasses a method for directing the expression of a therapeutic gene to be desirably expressed in certain squamous epithelium by the use of the medicament.